The exact cellular and molecular mechanisms involved in the pathogenesis of NTHi otitis media are not well understood. Several lines of evidence suggest that bacterial adherence (specific and non-specific interactions) and delivery of bacterial toxin on the target (epithelial) cell are critical for the pathogenesis of otitis media and the virulence factor. The paucity of such critical information makes it very difficult to formulate an effective approach to developing a vaccine or reduce the inflammatory response. The objectives of this project are to: define the cellular and molecular mechanisms involved in NTHi adherence on the respiratory epithelial cells; delineate the role of the endotoxin in the pathogenesis of otitis media; characterize the antigenicity to NTHi surface antigens as pathogenic and virulence factors; and characterize the role of mucosal immunity in protection and pathogenesis. A. Biological Properties of Released NTHi LPS: Release of lipopolysaccharide (LPS) from nontypeable H. influenzae (NTHi) may play an important role in the pathogenicity of otitis media caused by NTHi. The biological properties of the released lipooligosaccharide (LOS) showed a 16 to 37 fold increase in limulus amebocyte lysate (LAL) and a 13 to 28 fold increase in mouse lethal toxicity if compared with that of cell-bound LOS at the same concentration. The LAL activity of released LPS was also higher than that of purified LPS while both forms showed similar activity in the mouse lethal toxicity assay. In conclusion, NTHi released variable but significant amounts of LPS into the growth medium. This released LPS is more LAL active and more mouse lethal toxic than the cell-bound LPS. We are currently investigating cytokine levels from human peripheral blood monocytes stimulated by different LOS preparations. B. Study of bacterial adherence: The ultimate goal of this project is to isolate and clone the host receptor for the adhesion molecules expressed on the surface of the bacteria. To date, we have secured several human respiratory epithelial cell lines and rat middle ear epithelial cultures that can be used to test cellular and molecular mechanisms for the bacterial adherence. We are currently screening NTHi strains that are highly adherent to those cells.